Factor VIII: C (FVIII: C) is an essential blood coagulation factor, whose absence or loss of activity results in the clotting disorder Haemophilia A. Activated factor VIII: C acts as a cofactor to activated factor IX, which together activate factor X in the coagulation cascade.
Factor VIII: C (FVIII: C) is a plasma protein essential for blood coagulation whose deficiency or defective formation results in the blood clotting disorder known as Haemophilia A (Lenting et. al., 1998). FVIII: C is synthesized as a 300-kd precursor protein comprising of six domains: A1, A2, B, A3, C1 and C2. In the plasma, upon limited proteolysis by thrombin, it circulates as a heterodimer consisting of the heavy chain (A1-A2-B domains) and light chain (A3-C1-C2 domains) linked by a Calcium (II) ion through the A1 and A3 domains. This FVIII: C heterodimer circulates in the plasma as a non-covalent complex with von Willerbrand Factor (vWF), a large ˜220 kDa multimeric protein (Sadler, 1998). The association of FVIII: C with vWF results in its increased circulatory half-life, by avoiding premature proteolytic activation of factor VIII: C (Saenko et. al., 1999). While still in circulation, the heavy chain is further proteolysed where the B-domain is deleted. FVIII: C is activated by further proteolysis of the heavy chain resulting in a heterotrimer consisting of A1, A2 and A3-C1-C2 subunits (Shen et. al., 2008). Active FVIII: C (FVIII: Ca) associates with activated Factor IX (FIXa) to form the “X-ase” (ten-ase) complex which activates Factor X (FXa). FXa, along with activated Factor V (FVa), activates the prothrombinase complex that converts prothrombin to thrombin, resulting in a blood clot (Wang et. al., 2003). However, FVIII: Ca quickly loses its activity through one of the following mechanisms: (i) by spontaneous dissociation of the A2 subunit from the A1-A3C1C2 complex (Fay and Smudzin, 1992), or (ii) by proteolytic degradation by activated protein C, thrombin, FXa, FIXa, etc. (Fay, 2004). FVIII: Ca is thus quickly cleared from the system.
The light chain of factor VIII: C has sites for phospholipid binding and vWF binding in the C2 domain (Nogami et. al., 2007), while the heavy chain is primarily responsible for the correct orientation of the factor VIII: C light chain-heavy chain complex and also for binding to factor IXa through the A1 domain at residues in the region between amino acids 558-565 and residues near amino acid 712 (Ngo et. al., 2008), which is essential to provide coagulation activity.
Haemophilia A is caused by either absence or the loss of factor VIII: C activity, which could be due to a number of reasons, including genetic defects resulting in the improper folding/secretion of factor VIII: C (White and Shoemaker, 1989) or development of auto-antibodies against factor VIII: C (Shima, 2006). To counter the deficiency or the loss of factor VIII: C activity observed in haemophilia patients, FVIII: C is administered as plasma concentrates (Marchesi et. al., 1972), or as recombinant factor VIII: C expressed using mammalian cells (Wood et. al., 1984; Kaufman et. al., 1988). The purification of factor VIII: C from plasma-derived sources is limited by two major bottlenecks: the scarcity in obtaining sufficient amounts of plasma from healthy donors to purify factor VIII: C; and the risk of viral contamination, though methods to reduce the risk of viral contamination have evolved through solvent/detergent extraction methods (Mannucci, 2010). These considerations made recombinant factor VIII: C an alternate option for the treatment of Haemophilia A.
Recombinant factor VIII: C preparations were first made in 1980s (Wood et. al., 1984). Furthermore, it has been shown that the B-domain of FVIII: C is dispensable for its coagulation activity (Eaton et. al., 1986). A number of B-domain deleted factor VIII: C products like ReFacto®/Xyntha™ (Wyeth Pharma) are currently available in the market. However, this B-domain deleted factor VIII: C (BDD-FVIII: C) is still a large molecule of five domains thus making its expression difficult.
Conventional Haemophilia A therapy involves the infusion of either plasma-derived Factor VIII: C or recombinant Factor VIII: C expressed in mammalian expression systems. The scarcity and risk in obtaining plasma-derived Factor VIII: C, the difficulty in expressing full length recombinant Factor VIII: C demands newer methods and or alternate strategies for producing functional Factor VIIIC therapeutic molecule.
The present disclosure aims at overcoming the drawbacks of the prior art by making use of the Pichia pastoris expression system to generate functional factor VIII chains.